The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. These reactions play essential roles in numerous biological processes important to human health, including oxidative phosphorylation in mitochondria. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. A new method to study biological electron transfer has been introduced which utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(II)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of interprotein electron transfer. Photoexcitation of Ru(II) to a metal-to-ligand charge-transfer state, Ru(II*), leads to rapid reduction or oxidation of a redox center within 16 E. This new technique is being used to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome bc1, and cytochrome c peroxidase. A new ruthenium dimer has recently been developed which binds with high affinity to cytochrome bc1 and can photooxidize cyt c1 within 1 microsecond. This new technique has been used to measure the rate constant for electron transfer between the Rieske iron-sulfur center and cyt c1 for the first time. The specific aims for the next grant period are to: 1) Design new ruthenium complexes that maximize the rate and yield of photoreduction and photooxidation of biological redox centers. 2) Carry out a detailed study of electron transfer within cytochrome bc1. A major goal will be to determine what factors control the conformational changes in the Rieske iron-sulfur protein as it transfers electrons from QH2 in the Qo site to cyt c1. 3) Characterize electron transfer between cyt c and cyt c1 in the cytochrome bc1 complex. 4) Characterize electron transfer within the cytochrome c -- cytochrome c oxidase complex using rapid kinetics and site-directed mutagenesis. Major goals will be to determine the pathway and kinetics of electron transfer from cytochrome c through CuA and heme a to the heme a3--CuB binuclear center, and how electron transfer is coupled to proton pumping across the membrane. Biological electron transfer reactions play essential roles in numerous important biological processes, including oxidative phosphorylation in mitochondria. Defects in electron transfer proteins are responsible for a number of human health problems, including mitochondrial myopathies, aging, and degenerative diseases. In the proposed studies, new ruthenium photoexcitation methods will be developed to study electron transfer reactions important to human health.